β-1,4 Mannans are major components of hemicellulose in plant cell wall of softwood, plant seeds and beans. Four types of polysaccharides including linear mannan, galactomannan, glucomannan, galactoglucomannan that are linked via β-1,4-glycosidic bonds compose mannans. Mannan hydrolysis provides wide array of biotechnological applications, such as feed manufacture, pulp and paper industries, and hydrolyzing coffee extract to reduce viscosity. A set of enzymes are required for complete degradation of mannans, including endo-β-1,4-mannanase (β-mannanase, EC 3.2.1.78), exo-β-mannosidase (EC 3.2.1.25) to cleave the main chain, and β-glucosidase (EC 3.2.1.21), α-galactosidase (EC 3.2.1.22), and acetyl mannan esterase to remove side chain decoration. Among them, β-mannanase which catalyzes random hydrolysis of manno-glycosidic bonds in the main chain is the key enzyme. More recently, major products of β-mannanase, mannotriose and mannobiose (mannooligosaccharides, MOS), have been proved beneficial as animal nutrition additive due to its prebiotic properties.
β-Mannanases are derived from various organisms including bacteria, yeasts, and filamentous fungi. According to the amino acid sequence homology, β-mannanases are mostly classified to glycoside hydrolase (GH) families 5, 26 and 113. These families share the same (β/α)8 folding and catalytic machinery, that two glutamate residues at active site serve as general acid/base and nucleophile to catalyze the cleavage of glycosidic bonds via a retaining double displacement mechanisms. Since industrial process is usually carried out at high temperatures, stable enzyme usage under a broad range of temperature is highly desirable. Therefore, β-mannanase needs to be modified to meet the requirement for different industrial usages. There are two ways to achieve these goals, one way is to screen suitable genes in nature, and the second way is modifying current enzyme genes based on their 3-D structural information.
In the present invention, the crystal structure of β-mannanase is analyzed and the enzyme activity of β-mannanase is improved by site-directed mutagenesis of the gene.